PROJECT SUMMARY/ABSTRACT Classical immunogenetics, linkage studies, and genome-wide association studies (GWAS) have identified a set of ~50 chromosomal regions that individually and significantly contribute to the risk for type 1 diabetes (T1D). Knowledge of the genes in these regions provides an opportunity to probe the molecular underpinnings of the disease, potentially facilitating improved disease prediction and/or novel strategies for prevention or therapeutic intervention. ImmunoChip fine mapping in T1D indicates that the majority of credible causative genetic variants for T1D overlap with tissue specific enhancer (but not promoter) elements, emphasizing the need for a context dependent understanding of the mode of action for genes contributing to T1D pathogenesis (Projects 1 and 2) as well as through the study of relevant cell types; an emerging notion for this P01 renewal (and the focus of Project 2). To address this need, Project 3 has, in preliminary studies, processed more than 800 RNA-Seq samples; transcriptionally profiling T and B lymphocyte subsets in both T1D cases and controls. These efforts have yielded three key observations: 1) Genes located in T1D-associated chromosomal regions are significantly enriched for cell-type specific alternative splicing events. These alternative splicing events include intron retention or exon skipping activities that are likely to have significant effects on the expression and function of the proteins encoded by these genes. 2) Identification of non-coding polymorphisms in regulatory regions that are recognized as credible causative variants for T1D and are significantly associated with these alternative splicing events in a highly tissue-specific manner. 3) Genes located in chromosomal regions associated with T1D that display correlated expression in lymphocytes, allowing them be clustered into co-expression modules that provide insights into regulation and function. Our preliminary findings suggest that transcript isoforms and, by implication, the protein isoforms that they encode, are altered in T1D through genetically regulated shifts in exon usage in specific cell types. To gain a comprehensive understanding of the impact of alternative splicing and alternative transcript usage on T1D risk, and to identify and characterize disease relevant transcripts and isoforms, we propose to apply long read sequencing to RNA from selected lymphocyte populations in T1D and jointly analyze short and long read data to obtain reliable, cell type specific, information on transcript structure and usage. We will then characterize the downstream effects of alternative splicing and transcript isoform usage on a key T1D gene, IKZF4, which anchors a co-expression module in regulatory T cells. Finally, we will characterize the impact of T1D causative genetic variants on chromatin accessibility and DNA methylation at the T1D candidate genes they flank. These studies are highly synergistic with Projects 1 and 2 where those efforts propose to utilize clinical samples and isogenic cellular models to examine key genotype:phenotype associations in specific cell subsets.